CN110368526A - A kind of medical acellular organism material and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of medical repair materials, and relates to a medical decellularized material for tissue repair and a preparation method thereof. The decellularized material and preparation method of the present invention neither use any chemical cross-linking agent or optical method, nor use physical methods to make up for the problems of poor stability, easy degradation, and insufficient mechanical properties of existing materials, and avoid chemical reagents. residue and toxicity. The present invention selects the connective tissue of the breeding stock as the raw material, does not select the commercial meat stock as the source of the raw material, and the connective tissue of the breeding stock has stronger toughness and better biomechanical properties than that of the commercial meat stock. The decellularized medical material of the present invention not only retains the advantages of the existing decellularized materials, but also has better stability and mechanical properties, is not easy to break, is easy to suture and has good airtightness, and is especially suitable for requirements on stability and mechanical properties. Tissue repair at high tissue sites.

Description

A kind of medical decellularized biological material and its preparation method

technical field

The invention belongs to the field of medical biological materials, and relates to a medical decellularized biological material and a preparation method thereof.

Background technique

At present, biomaterials used in clinical medicine are divided into two categories: synthetic materials and natural biomaterials; synthetic materials have good mechanical strength, good stability, and are not easy to degrade, but they cannot induce tissue regeneration and healing. Natural biological materials are mostly decellularized materials, mainly derived from mammalian dermis, small intestine, pericardium and other tissues, which are processed by fat removal, cell removal, and DNA removal to obtain three-dimensional materials with extracellular matrix. The structure and some active ingredients, including collagen, non-collagen and growth factors, provide an adaptive environment for host cell adhesion, proliferation, and differentiation; contribute to the structural reconstruction of damaged tissues, and then promote tissue repair and regeneration and functional recovery.

The main problem encountered at present is that in order to ensure a high degree of safety for this kind of decellularized medical materials, it is necessary to ensure that there is no immune rejection and no risk of infection; in actual application production, in the production process of this kind of decellularized materials, Especially the process of decellularization is very harsh, and the processing time is long, sometimes reaching more than 10-20 hours. This kind of processing has different degrees of influence and damage on the tissue ECM, coupled with the reasons of the tissue raw materials themselves, leading to the present situation. There are decellularized medical materials, which have poor stability, rapid degradation, and insufficient mechanical strength of sheet-like finished products (called biological patches); in order to make up for this deficiency and overcome the defects of the finished products; During processing, chemical cross-linking agents such as epoxy or glutaraldehyde are additionally used, or shared with synthetic materials, in order to meet the mechanical performance requirements of the finished product and prevent excessive degradation. However, the use of chemical cross-linking agents may have the following disadvantages: potential cytotoxicity, and the degree of cross-linking is difficult to accurately control; excessive cross-linking will result in slower degradation rate, which does not match tissue regeneration, and can lead to fibrosis, chronic Inflammation and other reactions.

The Chinese patent with application number 201710862130.X, cruciate ligament regenerative implant and its preparation method and application, although it meets the mechanical requirements of tissue repair surgery, is made of degradable polymers and fibrinogen, etc. Biomaterials that guide tissue regeneration are blended and prepared by electrospinning technology.

The Chinese patent with the application number 201310203598.X, an artificial biological tissue and its preparation method, is simply curled or superimposed, and the force between the layers is very small. It is likely that the stress layer is uneven due to layering. A situation occurs in which the layers are broken separately.

An ideal decellularized medical material should meet the following conditions: ① It is safe, non-toxic, and non-infectious; ② It has good tissue compatibility and no immune rejection. ③Good compactness and no permeability. ④ If it is used for suturing, it must be tough, easy to suture firmly, and not easy to break. ⑤It can promote tissue regeneration without adhesion. ⑥Easy to use, simple, easy to disinfect and sterilize. ⑦ Wide range of materials and low price.

Regarding the mechanical properties of decellularized medical materials, the dermis, pericardium, peritoneum, tissue, and small intestine from commercial meat animals are often used as the initial raw materials of decellularized medical materials; It often fails to meet the mechanical requirements of decellularized medical materials; therefore, in the process of preparing decellularized medical materials, the following two methods are often used to enhance the mechanical properties of the patch and enhance the stability of the product to achieve good toughness and high tensile strength. Require.

One is: chemical (biochemical) method, in the patch preparation process, various cross-linking agents such as epoxy, glutaraldehyde, genipin, transglutaminase (TGase) are used to improve the stability of the patch. biomechanical properties. The disadvantage is that the degree of cross-linking is difficult to control, and sometimes chemical cross-linking agents may remain. These cross-linking agents are potentially cytotoxic, and this type of cross-linking patch usually has good mechanical properties, strong resistance, and is not easy to degrade or The degradation is slow, and the degradation and tissue regeneration cannot be matched synchronously, which can easily lead to adverse reactions such as fibrosis and chronic inflammation; at the same time, this kind of patch has sufficient mechanical strength, but the degradation is slow, which can lead to fibrosis and chronic inflammation; and because of its hardness , cannot fit perfectly with the tissue surface.

The second is: physical methods, such as placing the single-layer sheet horizontally or vertically, and overlapping the multi-layer cross parts. Although the patch prepared by this method as a whole has improved tensile strength, more raw materials for the patch are used. On the one hand, the raw materials are wasted, and on the other hand, the patch is too thick, which will delay the complete degradation of the entire patch for too long, thereby affecting the repair of the target tissue and the recovery of function. In addition, the raw material of the patch is made into thin strips and then twisted into a thread, and then the patch is made by weaving; the tensile strength of the patch made by weaving is obviously improved, but this step needs to be made of the patch raw material first. It is technically difficult to make thin strips and then prepared by weaving, and the steps are a bit complicated.

Contents of the invention

One of the purposes of the present invention is to solve the problem that some decellularized medical materials have poor stability, are easy to degrade, or degrade quickly in actual clinical use, and cannot effectively meet the actual needs of medical clinics. For example, the product degrades too quickly, which is not conducive to As a reliable and stable cell scaffold, it assists the formation of new tissue and the recovery of function at the injured site.

The second object of the present invention is to solve the problem that some decellularized medical materials have poor and weak mechanical properties in actual clinical use, and are prone to rupture, tearing or other accidents in actual clinical use, which cannot effectively meet the needs of medical clinical practice. For example, hernia mesh, tendon mesh, rotator cuff mesh, and dura mater mesh with poor mechanical properties will appear bulging, tearing or cracking.

The third purpose of the present invention is to avoid the above-mentioned shortcomings and defects in the methods and products that may exist in existing medical materials such as cross-linking agents and weaving methods to enhance the stability and mechanical characteristics of decellularized materials.

Focusing on the specific technical characteristics of material stability and biomechanics, the inventor has read a large number of documents, combined with his own in-depth research and careful analysis of academic theories, and many years of rich work experience to closely combine theory with practice, which is ingenious and convenient The technical problems of insufficient stability and weak mechanical properties of decellularized medical materials have been completely solved.

The main innovation point of the present invention is that the tissue or organ of breeding stock is selected as the raw material for decellularization instead of commercial meat stock.

Because the existing animal-derived decellularized raw materials are taken from freshly slaughtered animals and processed through decellularization processes, the freshly slaughtered animal tissues used are all commercialized pork pigs, beef cattle, and mutton sheep that have just been slaughtered; Most of the pigs waiting to be slaughtered in the slaughterhouse are commercialized meat pigs that have just come out of the slaughterhouse, and the outer three-way hybrids, such as Du Dachang, are the main breeds, and there are also some inner three-way meat pigs; the weight of the slaughtered pigs 90-120 kg; the feeding age is 5-6 months. According to the season and the price of pigs, the weight and days of slaughtering pigs vary.

The inventor's technical idea to solve the problems of poor stability, easy degradation, and weak mechanical properties of the product in decellularized medical materials is not to stay in the slight improvement of the physical method when processing the decellularized materials, such as which method is used to overlap (longitudinal /horizontal/oblique), what is the overlap ratio, how many layers need to be overlapped, the thickness of the raw material thread used in weaving, whether it is plain weave, twill weave or twisted weave, etc. Concentration improvement, optimization of cross-linking time and other technical parameters; instead, start from the source of raw materials, the first step in the preparation of decellularized biomaterials, starting from the initial animal source, and use this as a technical breakthrough point or focus to carry out in-depth research Research shows that the main innovation point of the product of the present invention lies in the selection of animal tissue raw materials for decellularized materials. It is not the commercial-grade meat animals that everyone uses now, but the breeding animals of the prime of life, preferably middle-aged and mature sows and breeding cows. , preferably healthy dams are culled due to decreased reproductive performance.

The inventor went deep into the front line of pig raising, and after fully communicating with the person in charge of a large pig farm, according to the breeding mode (usually all self-propagation and self-raising) and the breeding cycle (the pigs will be slaughtered within 5-6 months) , with a body weight of about 90-120 kg; and the sows were gradually eliminated after eight to ten births due to the decline in reproductive performance. At this time, they have been raised for more than 36 months and their body weight has reached 180 kg). Tissues from different parts (dermis, small intestine submucosa) of sows and commercial pigs were taken, and finished materials were prepared by basically similar material preparation methods (including the same decellularization process, etc.); then the mechanical properties of these finished materials were tested respectively. It is found that the tensile strength (i.e. toughness) of various materials derived from sows is significantly higher than that of the same parts of commercial pigs; in addition, organs or tissues from culled sows are used to prepare medical Cell-free biomaterials also have a very obvious price advantage. The price of the same tissue parts, the price of culled sows, is lower than that of commercial meat pigs, for example, the same small intestine, the price of culled sows is 30-50% lower; Selecting corresponding tissue parts from culled sows, such as small intestine, dermis, etc., as raw materials for decellularization can be said to be of high quality and low price; kill two birds with one stone.

The secondary innovation of the product of the present invention is that the preferred tissue site is the small intestinal submucosa (SIS), and the dermis can also be selected; because the materials made from decellularized tissues of this type all contain natural three-dimensional structures, but are different from other tissues. In terms of site comparison, the composition of the small intestinal submucosa (SIS) is mainly composed of type I fibrous collagen, and also contains type III, IV, and VI collagen, especially the important type IV collagen, which is beneficial to new blood vessels and The formation of the basement membrane has a significant promoting effect; in addition, according to many articles published by Dr. STEPHEN F. BADYLAK in the United States, the degradation products of SIS patch have strong antibacterial activity, which is similar to porcine defensin (pBD-1) ) function; and GeorgeS.Hussey's 2018 article "Extracellular Matrix Bioscaffolds for Building Gastrointestinal Tissue" reported that SIS patch degradation products also have the effect of inducing cell chemotaxis and mitosis.

Invention principle:

For the decellularized medical material of the present invention, the source of the decellularized tissue raw material is breeding stock, for example, a sow that has given birth to several litters usually weighs significantly more than commercial pork pigs, and its volume is also significantly larger; some adult multiparous Sows can reach 200 kg, and the feeding time is usually more than 24 months. The age of culled sows may reach 40 months or more, and the weight may exceed 200 kg, and some individuals will reach 220-250 kg. Compared with commercial pigs, the tissues and organs of adult sows are fully developed in all aspects, and they have achieved full and comprehensive maturity in the true sense in terms of sub-organ level and structure, mesoscopic and microscopic properties, and histological strength. ; further from the molecular level and molecular structure, it is possible that the degree of hydroxylation of collagen is high, the stability of the triple helix structure is better, the degree of natural cross-linking between collagens is high, the stability is good, and it is not easy to degrade; usually the cross-linking bonds of collagen It is formed by the amino group and aldehyde group of lysine or hydroxylysine, under the action of lysyl oxidase, undergoes oxidative deamination reaction, and the covalent bond of the obtained lysine formula is very stable; macroscopically Because the mechanical properties of the connective tissue are better, the prepared material has a high tensile strength detected.

However, the current decellularized materials are derived from commercial meat animals, such as meat pigs or beef cattle, which usually have a short feeding period; —120 kg, which is obviously lighter than the weight of 180-200 sows or more (according to breed and parity, the weight of sows is different), which is only about half of its weight; at the same time Compared with reproductive sows, the tissues and organs of commercial meat animals only take shape on the surface, and the frame structure is formed, but in fact, their internal structure, microscopic performance, histological strength and other indicators have not been fully and comprehensively analyzed. Mature, further from the molecular level and molecular structure, it may be that the degree of hydroxylation of collagen is low, the stability of the triple helix structure is poor, the degree of natural cross-linking between collagens is low, the stability is poor, and it is relatively easy to be degraded; macroscopically, it is manifested as connective The mechanical properties of the tissue are poor, and the tensile strength of the prepared material is low.

Compared with the prior art, the present invention has the following significant advantages and beneficial effects:

1. The invented decellularized medical material product uses breeding stock tissue as raw material, has good natural cross-linking degree, good stability, and is not easy to degrade. The material made has good toughness, high tensile strength, not easy to break, and wear resistance it is good;

2. The invented decellularized medical material product uses the small intestinal submucosa and dermis as raw materials, retains the three-dimensional structure of the extracellular matrix, various functional proteins, growth factors, and glycosaminoglycans, and has a good ability to induce tissue regeneration Function, can accelerate the growth of postoperative tissue and reconstruction of function;

3. The invented decellularized medical material product has no cross-linking agent in production, no cross-linking agent residue, no potential cytotoxicity, and will not cause fibrosis or chronic inflammation;

4. The decellularized medical material product invented has an appropriate degradation rate, will not degrade too quickly, and the degradation rate will be slower; at the same time, its ECM three-dimensional structure has the function of inducing cells and blood vessels to grow in, and it will gradually grow into itself when new tissue grows in. degradation.

In addition, from an economic point of view, the raw material can be multiparous sows that have been eliminated due to the decline in reproductive performance. Usually, the price of such raw materials is significantly lower than that of commercial pork pigs; for future large-scale industrial production, it is necessary to purchase raw materials in large quantities , Using culled sows as the raw material for decellularization can save a lot of expenses for the enterprise, which is also one of the innovation points.

The purpose of the present invention is to provide a decellularized medical material and a preparation method thereof. The purpose of the present invention is achieved through the following technical solutions.

The tissue raw material of the decellularized medical material is derived from adult breeding stock instead of commercial meat stock.

Further, the adult breeding stock refers to adult breeding pigs, breeding cattle, sheep, horses, camels, donkeys, mules and other artificially raised mid-to-large mammals that are bred mainly for breeding offspring.

Further, the adult breeding stock refers to multiparous sows, cows, ewes, mares, female camels, female donkeys, female mules and other multiparous artificially raised medium and large mammals.

Further, preferably, the adult breeding animals are sows, cows, ewes, mares, female camels, female donkeys, female mules, etc. that have been eliminated due to reduced reproductive performance.

Further, the raw material of the decellularized medical material includes one or a combination of raw materials of small intestine submucosa, dermis, visceral membrane, peritoneum, and pericardium of breeding stock.

Further, the raw material of the decellularized medical material is small intestinal submucosa and dermis of culled sows and cows.

Further, the decellularized medical material is processed through processes such as pretreatment, disinfection, fat removal, cell removal, DNA removal and α-Gal antigen removal, and drying and sterilization; it can also be selected according to the target physical state of the finished product , using physical, chemical or biological (such as enzyme) methods to process into corresponding states, such as solid flakes, granules, gels, and then sterilized.

The present invention also provides a method for preparing decellularized medical materials, which mainly includes the selection of raw materials in the first step, that is, selecting the corresponding tissue parts of the breeding stock as the raw material for decellularized products. The specific preparation method of the product includes:

1) Take materials and wash: take the connective tissue of the breeding stock and wash it thoroughly;

2) Pretreatment, pre-disinfection, and degreasing are processed in three steps;

3) Decellularization: use a solution or mixed solution made of surfactant, protease, EDTA or other decellularization reagents, soak the decellularized raw materials under ultrasonic conditions, and then rinse with PBS;

4) To remove DNA and remove α-Gal antigen: Soak the decellularized raw materials in an aqueous solution containing DNase, after washing, soak in an aqueous solution containing α-galactosidase; you can also use 10--50mM alkaline solution, ultrasonic Under the condition, soak; then use PBS ultrasonic cleaning until neutral;

5) The semi-finished product is made into a corresponding physical state as required, and then processed into a finished product through steps including drying, packaging, and sterilization.

Further specific operation methods are:

1) Pretreatment, sampling and cleaning: take the small intestine or skin tissue of the breeding stock, and fully wash; remove the non-connective tissue in the small intestine and skin tissue by mechanical scraping or shearing; separate the submucosa or dermis of the small intestine ;Rinse clean, the obtained target tissue is the raw material; then soak it in acetic acid solution for 30--120 minutes, the ratio of raw material to acetic acid solution is 1:5--1:10 (W/W) ;

2) Disinfection: Use a mixed solution containing peracetic acid and ethanol to soak the raw materials under ultrasonic and room temperature conditions for disinfection; the concentration of peracetic acid is 0.5-1.5%, and the concentration of ethanol is 15-25%. The ratio of the mixed aqueous solution is 1:5--1:10, and the soaking time is 30--120 minutes; then ultrasonic cleaning with purified water;

3) Degreasing: Use alcohol solution to soak the material raw materials under ultrasonic and normal temperature conditions. The ratio of material raw materials to alcohol solution is 1:5-1:10, and the soaking time at room temperature is 0.5-6h; after that, ultrasonic cleaning with water;

4) Decellularization: use a mixed solution containing 0.05M protease and EDTA, soak the raw materials under ultrasonic and low temperature conditions, and then rinse with PBS; repeat 5 steps 1-3 times;

5) Remove DNA and remove α-Gal antigen: Soak the raw materials in an aqueous solution containing DNase, after cleaning, soak the raw materials in an aqueous solution containing α-galactosidase, and then use 10--30mM NaOH aqueous solution at room temperature 1. Under ultrasonic conditions, soak the raw materials; then ultrasonically clean with PBS until neutral;

6) Make the semi-finished material into the desired physical state, then dry, pack and sterilize to obtain the finished product.

Chinese and English terms/nouns are firstly understood according to the following text descriptions, unless otherwise specified in detail; other terms are understood according to the level of ordinary skilled in the art.

Explanation of terms in Chinese and English:

1) Breeding stock: it is for commercial meat animals; the main purpose of breeding is to breed young animals, not as short-term breeding of commercial meat animals (for example, the breeding time of pork pigs is about 5-6 months); The main purpose of meat animals is to grow meat for supply; breeding animals include pigs, cattle, sheep, horses, donkeys, camels, and dogs; the breeding animals referred to in this patent are all adult, capable of reproduction and fertility, usually Breeding time is at least one year; for example, breeding pigs include sows and boars, and sows here refer to adult sows capable of reproduction, excluding young gilts;

2) Multiparous dams: part of the breeding stock, they are dams that have given birth to at least one litter; they are relative to back-up dams, which mainly refer to those that have not yet conceived because of their age or age young dams;

3) Elimination of female animals: This is part of the breeding stock; in the farm, the manager considers that when the reproductive performance of the female animal drops to a certain level, such as small litter size, low lactation, light weight of the weaning litter, and comprehensive consideration of the female pen The utilization rate of sows, the price of culled sows and other factors; Judging and analyzing from an economic point of view, it is believed that the value obtained by continuing to raise the sow has been significantly reduced, and it will be more cost-effective to eliminate such sows; There are certain differences in the judging criteria; for example, usually sows will gradually be eliminated after giving birth to 8 litters; there are slight differences between different breeds of pigs;

4) Tissue repair materials, tissue regeneration materials, bio-patch, bio-patch, bio-scaffold, degradable patch, absorbable patch Bio-Mesh, Bio-Patch, Patch, Bioscaffold, these Chinese and English terms or terms, Although they are different on the surface, their purposes and uses are basically the same; unless otherwise specified, the connotations of the above-mentioned terms are essentially the same;

5) Small Intestinal Submucosa SIS (Small Intestinal Submucosa): The small intestinal tissue includes the jejunum and ileum, and the remaining part after removing the small intestinal mucosal layer, muscular layer, and serosa layer is mainly composed of collagen, accounting for more than 80% ;

6) Dermis: It is a part of the skin, between the epidermis and the subcutaneous layer; differentiated from the mesoderm, it is divided into two layers, the papillary layer and the reticular layer, and there is no obvious boundary between the two. The dermis is generally 1 to 2 mm thick; it is mainly composed of fibroblasts and the collagen fibers and matrix produced by them, and has blood vessels, lymphatic vessels, and nerves attached. The matrix is composed of a complex of mucopolysaccharides and proteins such as hyaluronic acid and chondroitin sulfate, which fills the gaps between collagen fibers and fiber bundles and between cells, and is hydrophilic;

7) Extracellular matrix ECM (Extracellular Matrix): It is a non-cellular component that exists in all tissues and organs. It not only provides the necessary physical support for cell tissues, but also provides a suitable place and microscopic It also plays an important role in regulating the morphogenesis of tissues, cell chemotaxis and differentiation, and important physiological, biochemical and biomechanical aspects, thereby affecting or regulating the functions of tissues and organs. 50% of the function of cells is determined by the external microenvironment created by the extracellular matrix.

Unless the context clearly dictates otherwise, the singular forms "a" and "the" used in this specification and the appended claims include plural reference. Thus, for example, "the method" includes one or more methods, and/or steps, They are of the type described herein and/or would be apparent to those skilled in the art after reading the text. The term "about" or "approximately" refers to a range of values in the statistical sense, and the range may be within an order of magnitude Within, usually within 50% of the specified value or range, further within 20%, more usually within 10%, even more usually within 5%. The term "about" or "approximately" covers the permissible Variations depend on the particular system studied and are readily recognized by those skilled in the art.

Below in conjunction with specific embodiment, to further describe the principle and scheme of the present invention; It should be understood that these embodiments are only for illustration and convenience to understand the thought of the present invention, but can not be limited thereto; Embodiment does not limit the present invention in any way Scope, in the following examples, various processes and methods that are not described in detail are methods of common knowledge used in this field; it may also include omitting common methods and methods used in the field of medical biomaterials.

Detailed ways

Example 1: (elimination of sow small intestine)

Select the preparation of decellularized medical materials derived from the small intestinal submucosa of healthy culled sows, and the specific steps are as follows:

1) Pretreatment: In the slaughterhouse, the fresh small intestines of culled sows are selected, cleaned and used as the initial raw material for the patch; after in-depth research, the gestational age of culled binary mixed sows is usually above 6 parities, and the weight is 180 kg The above are all in the empty pregnant stage; the feeding time is usually more than 36 months; the mucosal layer, muscular layer, serosa layer, lymph nodes, etc. of the pig small intestine and jejunum are removed by physical scraping, the submucosa is separated, and soaked in 0.5% acetic acid solution for 30 Minutes, the ratio of pig small intestine to acetic acid solution was 1:5, and then rinsed with purified water 3 times to obtain the raw material of the biological patch, namely the small intestinal submucosa, hereinafter referred to as SIS material;

2) Disinfection: use a mixed aqueous solution containing 1.0% peracetic acid and 15% ethanol, the ratio of the SIS material to the mixed aqueous solution is 1:10, and immerse at room temperature for 100 minutes under ultrasonic conditions for disinfection. Then use purified water to ultrasonically clean 3 times;

3) Degreasing: Use ethanol with a concentration of 90%, the ratio of SIS material to ethanol is 1:10, soak at room temperature for 2 hours under ultrasonic conditions. Then use water for injection to ultrasonically clean 3 times;

4) Decellularization: use a mixed aqueous solution containing 0.02% trypsin and 0.02% EDTA, the ratio of SIS material to trypsin/EDTA solution is 1:5, soak for 30 minutes under ultrasonic conditions, and then soak twice in PBS solution ; Repeat the aforementioned decellularization step twice;

5) Remove DNA and remove α-Gal antigen: Use an aqueous solution containing 5U/ml DNase, the ratio of SIS material to DNase solution is 1:5, soak at 37°C for 20 minutes under ultrasonic conditions; then use PBS Rinse 3 times; use an aqueous solution containing 5U/ml α-galactosidase, the ratio of SIS material to α-galactosidase solution is 1:5, and soak at 30°C for 20 minutes under ultrasonic conditions; then use Soak in PBS solution twice; then use NaOH aqueous solution with a concentration of 10mM, the ratio of SIS material to NaOH solution is 1:20, and soak for 50 minutes at room temperature under ultrasonic conditions. Then use PBS ultrasonic cleaning until neutral;

6) Shaping, freeze-drying, and sterilization: four sheet-shaped raw materials after decellularization are overlapped and fixed on the mold, after freeze-drying, packaged, and finally irradiated and sterilized.

Example 2: (Commercial Pork Small Intestine Control Group 1)

The pretreatment of connective tissue, disinfection, degreasing, decellularization, DNA and α-Gal antigen removal, freeze-drying, sterilization and other steps are completely the same as in Example 1; the difference is only in the initial animal selection; selection of slaughter In the farm, the fresh small intestine tissue of the Du Chang Sanyuan miscellaneous commodity pork pig with a weight of about 100 kg was cleaned; it was learned that the number of days of breeding was about 160-180 days.

Example 3: (removal of sows --- leather)

The pretreatment of tissue raw materials, such as disinfection, degreasing, cell removal, DNA removal and α-Gal antigen removal, freeze-drying, and sterilization, are basically the same as in Example 1; but there are differences in the first step and the sixth step; the first step The difference is the different types of raw materials. In this example, skins from healthy culled sows (live pigs weighing more than 180 kg) from slaughterhouses were used as the initial raw materials. treatment, and remove the stratum corneum and epidermis, and use mechanical methods to make the thickness of the dermis 0.5mm, so as to facilitate subsequent treatments.

The second difference is that in the sixth step, the decellularized sheet material is divided into two pieces, instead of four layers like SIS; the overlapping is fixed on the mold, after freeze-drying, packaging, and finally sterilization.

Embodiment 4: (Commercial pork pig leather control group 2)

The pretreatment of tissue raw materials, such as disinfection, degreasing, cell removal, DNA removal and α-Gal antigen removal, freeze-drying, and sterilization, are basically the same as in Example 1; but there are differences in the first step and the sixth step; the first step The difference is that the types of raw materials are different. In this embodiment, the skin of healthy commercial pork pigs (live pigs weighing about 100 kg) from the slaughterhouse is used as the initial raw material. treatment, and remove the stratum corneum and epidermis, and use physical methods to make the thickness of the dermis 0.5mm, so as to facilitate subsequent treatments.

The second difference is that in the sixth step, the decellularized sheet material is divided into two pieces, instead of four layers like SIS; the overlapping is fixed on the mold, after freeze-drying, packaging, and finally sterilization.

Example 5: Testing of mechanical properties of decellularized medical materials.

The tensile strength of the decellularized medical material samples prepared in Examples 1-4 was tested.

Method: Cut the sample into a shape with a width of 10mm along two directions; after cutting, place it in an environment with a relative humidity of 40%-60% and a temperature of 22±2°C for 2 hours before testing. The distance between the clamps is 25 mm. Fix the two ends of the sample on the chuck of the tensile testing machine, stretch at a speed of 100 mm/min, and record the maximum force value at the time of breaking.

The results are shown in the table below:

Those skilled in the art can make various simple changes or adjustments or combinations to the present invention according to the above description; therefore, without violating the claims of the present invention, some details in the embodiments should not constitute For the limitation of the present invention, the present invention shall take the scope defined by the appended claims as the scope of protection.

Claims (6)

1. a kind of medical acellular organism material, which is characterized in that the acellular organism material, with the tissue of breeding stock, organ Or combinations thereof object be primary raw material.

2. biomaterial according to claim 1, which is characterized in that the breeding stock is, sow, cow, ewe through producing, One of mare, female camel, jenny ass, female mule or combination.

3. biomaterial according to claim 1, which is characterized in that the breeding stock is superseded healthy sow, cow.

4. biomaterial according to claim 1, which is characterized in that the organ of the breeding stock be liver, pancreas, heart, One of kidney, lungs, tracheae or combination.

5. biomaterial according to claim 1, which is characterized in that the tissue of the breeding stock is submucous layer of small intestine, stomach One kind of submucosa, corium, pericardium, internal organs film, peritonaeum, pericystium or combinations thereof object.

6. a kind of preparation method of medical acellular organism material, which is characterized in that with the tissue of breeding stock, organ or combinations thereof object For primary raw material, cell technique is gone to be process.